Development and characterization of potent and specific peptide inhibitors of p60c-src protein tyrosine kinase using pseudosubstrate-based inhibitor design approach

The cytoplasmic protein p60(c-src), an ubiquitous nonreceptor protein tyrosine kinase (PTK) is a potential anticancer target as it is over-expressed and/or constitutively active in several cancer types. In addition, the phenotype of c-src knock-out mice is consistent with osteopetrosis, which suggests that inhibitors against this enzyme may also be therapeutic for osteoporosis. Using a known peptide substrate for c-src, MIYKYYF, as a template, we have developed a series of pseudosubstrate-based peptide inhibitors. Structure-activity relationship studies have been performed on one of these inhibitors, CIYKYYF. In a kinase assay using YIYGSFK as the substrate, CIYKYY has been demonstrated to inhibit p60(c-src), with an IC50 of 0.6 muM. Further truncation has led to the determination that even the smaller peptide, CIYK, is a moderately potent inhibitor with IC50 of 15 muM. Some improvement in inhibitory potency (IC50 = 11.8 mum) has been observed with the replacement of Tyr(3) in CIYK with beta-phenylalanine (beta-Phe). The tetrapeptide CI(beta-Phe)K will be used as a lead compound for future development of peptidomimetics and small molecule inhibitors that have the capacity to penetrate the plasma membrane of intact cells.